Structure of the biliverdin cofactor in the Pfr state of bathy and prototypical phytochromes

Abstract

Phytochromes act as photoswitches between the red- and far-red absorbing parent states of phytochromes (Pr and Pfr). Plant phytochromes display an additional thermal conversion route from the physiologically active Pfr to Pr. The same reaction pattern is found in prototypical biliverdin-binding bacteriophytochromes in contrast to the reverse thermal transformation in bathy bacteriophytochromes. However, the molecular origin of the different thermal stabilities of the Pfr states in prototypical and bathy bacteriophytochromes is not known. We analyzed the structures of the chromophore binding pockets in the Pfr states of various bathy and prototypical biliverdin-binding phytochromes using a combined spectroscopic-theoretical approach. For the Pfr state of the bathy phytochrome from Pseudomonas aeruginosa, the very good agreement between calculated and experimental Raman spectra of the biliverdin cofactor is in line with important conclusions of previous crystallographic analyses, particularly the ZZEssa configuration of the chromophore and its mode of covalent attachment to the protein. The highly homogeneous chromophore conformation seems to be a unique property of the Pfr states of bathy phytochromes. This is in sharp contrast to the Pfr states of prototypical phytochromes that display conformational equilibria between two sub-states exhibiting small structural differences at the terminal methine bridges A-B and C-D. These differences may mainly root in the interactions of the cofactor with the highly conserved Asp-194 that occur via its carboxylate function in bathy phytochromes. The weaker interactions via the carbonyl function in prototypical phytochromes may lead to a higher structural flexibility of the chromophore pocket opening a reaction channel for the thermal (ZZE → ZZZ) Pfr to Pr back-conversion.

Keywords: Computer Modeling; Infrared Spectroscopy; Photoreceptors; Phytochrome; QM/MM Calculations; Raman Spectroscopy; Resonance Raman; Spectroscopy.

FIGURE 1.

Structure and atom numbering of the biliverdin chromophore in PaBphP in the 3²-linked attachment to the protein. Inset, chromophore structure at ring A in case for 2(R),3(E)-PΦB type binding (π-electron rearrangement).

FIGURE 2.

Optimized structure of the chromophore binding pocket of the Pfr state of PaBphP according to the QM/MM model of lowest energy (QM/MMmin model).

FIGURE 3.

Evolution of the r.m.s.d. for the Cα atoms of PaBphP during the 6-ns MD simulation.

FIGURE 4.

Experimental RR and calculated Raman spectra of the Pfr state of PaBphP in H₂O (black) and D₂O (red) between 1200 and 1750 cm⁻¹. A, experimental RR spectrum; B, sum of the QM96/MM-calculated Raman spectra obtained from various snapshots of the MD simulations; C, calculated Raman spectrum for the QM/MMmin model.

FIGURE 5.

Experimental RR and calculated Raman spectra of the Pfr state of PaBphP in H₂O (black) and D₂O (red) between 600 and 1200 cm⁻¹. A, experimental RR spectrum; B, sum of the QM96/MM-calculated Raman spectra obtained from various snapshots of the MD simulations; C, calculated Raman spectrum for the QM/MMmin model.

FIGURE 6.

Superposition of the minimum energy structure of PaBphP bonded to a BV-type chromophore in the ZZEssa conformation (orange) and the structure of PaBphP bonded to a 2(R),3(E)-PΦB chromophore (cyan) (A) and the structure PaBphP with BV in a ZZZssa conformation (cyan) (B).

FIGURE 7.

A, calculated Raman spectrum of the Pfr state of PaBphP with a BV-type chromophore binding in the ZZEssa configuration (sum of snapshots, as in Figs. 4 and 5); B, experimental RR spectrum of the Pfr state of PaBphP; C, calculated Raman spectrum of the Pfr state of PaBphP with PΦB-type chromophore binding in the ZZEssa configuration (sum of snapshots); D, calculated Raman spectrum of the Pfr state of PaBphP with a BV-type chromophore binding in the ZZZssa configuration (single snapshot). The left panel displays the overview spectra, highlighting the most structure-sensitive spectral regions. The right panel is an expanded view of the C=C stretching region.

FIGURE 8.

Experimental RR spectra of the Pfr states of PaBphP (A) and Agp2 in H₂O (black) and D₂O (red) (B).

FIGURE 9.

Homology model for the Pfr state of Agp2. Bold letters refer to Agp2-specific amino acids compared with PaBphP.

FIGURE 10.

Experimental RR spectra of the Pfr states (in H₂O) in the C=C stretching (right) and HOOP region (left) of PaBphP (A), Agp2 (B), Agp1 (C), Agp1-Δ18 (D), Agp1-C20A (E), and CphB (F).

FIGURE 11.

Experimental IR and RR spectra of Agp1 in H₂O. A, IR difference spectra “Pfr minus Pr”; B, RR difference spectra “Pfr minus Pr”; and C, RR of the Pfr state. Black lines and numbers refer to Agp1 reconstituted with the unlabeled chromophore, and red lines and numbers refer to the chromophore ¹³C-labeled at position C-10; the shaded area indicates Pr and protein difference bands.

FIGURE 12.

RR spectra in the C=C stretching region of Agp2 (A), Agp1 reconstituted with the unlabeled chromophore (B), and Agp1 reconstituted with the chromophore ¹³C-labeled at position C-10 (C). The spectra were measured from protein solutions in H₂O. The dotted lines refer to fitted Lorentzian line shapes. Band components originating from modes of similar character are highlighted by different colors.

FIGURE 13.

Temperature dependence of the RR spectra of the Pfr state of full-length Agp1. Residual Pr contribution of the Pfr state was manually subtracted using the raw Pr spectrum at each temperature. Subsequently, a minimum set of Lorentzian functions was fitted to the spectra. For the sake of clarity, only the two curves describing the HOOP modes are shown (dotted lines). Only restricted variations of the frequencies and bands widths (≤1 cm⁻¹) were allowed in the global fit. The sum of the individual Lorentzians is essentially indistinguishable from the experimental spectrum (straight line). In an alternative approach, the fitting procedure was applied to the raw spectra by including band components originating from the Pr state determined before. Both fitting procedures afforded a very similar temperature dependence of the intensity ratio of the two HOOP modes, although there was a systematic underestimation of the lower frequency component in the latter approach (∼20%).

FIGURE 14.

RR spectra of the Pfr state of full-length Agp1 measured at −140 °C (A) and 20 °C (1064 nm excitation) (B).

FIGURE 15.

Plot of the logarithm of the equilibrium constant K for the conformational equilibrium in the Pfr state of Agp1 against 1/T. The equilibrium constant is defined by the intensity ratio of the low frequency (∼800 cm⁻¹) to the high frequency (∼809 cm⁻¹) band component of the prominent peak in the HOOP region. The intensities were determined by band fitting as described in Fig. 13.